Generally, L-tyrosine phenol-lyase is a ubiquitous enzyme produced by Erwinia sp. Escherichia sp. and Gram-negative intestinal bacteria which are cultivated in medium containing tyrosine as an inducer.
L-tyrosine phenol-lyase catalyzes the α, β-elimination reaction which hydrolyzes tyrosine into phenol, pyruvate and ammonia. It also catalyzes the tyrosine synthesis reaction which is reversible to the α, β-elimination reaction (see Scheme 1). When catechol, pyruvate and ammonia are provided, L-tyrosine phenol-lyase synthesizes a valuable amino acid, L-DOPA.
Scheme 1
On the other hand, L-tryptophan indole-lyase catalyzes the α, β-elimination reaction which hydrolyzes L-tryptophan into indole, pyruvate and ammonia. However, if ammonium is present at a high concentration in the medium, L-tryptophan is synthesized from indole and pyruvate by reverse α, β-elimination (synthesis) reaction (see Scheme 2).
Scheme 2
As mentioned above, L-tyrosine phenol-lyase and L-tryptophan indole-lyase are induced by L-tyrosine and L-tryptophan as inducers in the medium, respectively.
It has been reported that the valuable medico-amino acid, L-DOPA is effective in treating Parkinson's disease. Generally, L-DOPA is produced by enzymatic reaction using L-tyrosine phenol-lyase. An enzymatic biotransformation process of L-DOPA production generally uses an enzyme derived from mesophile. The enzyme stability in the enzymatic biotransformation process of L-DOPA production plays a key role in increasing the productivity and production rate. However, the enzyme derived from mesophile has low-stability to heat, so it is easily denatured. In addition, because it is very unstable in extreme conditions of processes using solvents, high pressure, extreme acidity or alkalinity, the productivity and production rate of the enzymatic biotransformation process is decreased.
To overcome these problems of enzymes derived from mesophile, we made an effort to develop technology using an enzyme derived from a thermophile in an enzymatic biotransformation process. Generally, a thermophile adapted to a high-temperature environment has thermostable enzymes stable to heat. Since the thermostable enzymes have a stability to solvents, a high concentration of hydrogen ions or chemical detergents as well as a stability to heat, we propose the possible industrial use of thermostable enzymes in an enzymatic biotransformation process (KR Patent No 0229284).
To discover a novel thermostable L-tyrosine phenol-lyase and L-tryptophan indole-lyase that could be used for the above enzymatic biotransformation process of L-DOPA and L-tryptophan production, the present inventors have developed a pure culture method of symbiotic bacteria. Using the pure culture method, these inventors have identified a novel obligately thermophile Symbiobacterium toebii SC-1 showing a high activity of thermostable L-tyrosine phenol-lyase and L-tryptophan indole-lyase.
In addition, the present inventors have developed a method of growth measurement for Symbiobacterium toebii SC-1 showing a low-growth yield, and a screening method of its relative symbiotic bacteria using a specific antibody to it.